RNAi COMPOUND TARGETED TO THROMBOSPONDIN-1 AND APPLICATIONS THEREOF

ABSTRACT

The present invention relates to an RNAi compound and an expression plasmid for inhibiting expression of Thrombospondin-1, which comprises a target sequence selected from Thrombospondin-1 gene. The present invention also related to a pharmaceutical composition comprising the RNAi compound and applications thereof. The RNAi compound can reduce the expression of Thrombospondin-1 to activate immune responses. In addition, the present invention also disclosed that an RNAi compound targeted to Thrombospondin-1 gene can delay tumor progression.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an RNAi compound and an expressionplasmid for inhibiting expression of Thrombospondin-1, a pharmaceuticalcomposition containing the RNAi compound, and a method of treatingcancer using the RNAi compound.

2. Description of Related Art

The Thrombospondin protein family consists of thrombospondin 1-5.Thrombospondin-1 (TSP-1) is widely distributed in normal tissue,including heart, lung, liver, spleen and stomach. TSP-1 is mainlysecreted by platelets and dendritic cells (DCs), and has multiplebiological functions including inhibition of angiogenesis, apoptosis,and activation of transforming growth factor beta (TGF-β) and immuneregulation.

Dendritic cells play a major role on activating adoptive immuneresponses and express TSP-1 during its inactivation. Then, the expressedTSP-1 converts the TGF-β from a latent state into an activated state.The TGF-β is a regulation factor of the immune system, and convertseffect T cells into regulatory T cells to suppress immune responses.TSP-1 also interacts with CD47 on T cells to enhance the apoptosis of Tcells and the formation of regulatory T cells, and inhibit the immuneresponses resulting from inflammation. Hence, the immune responses canbe improved if the expression of TSP-1 is inhibited.

Conventional immune therapy is accomplished by inducing an immuneresponse with abundant tumor-associated antigens. However, abundantinhibitory cytokines are also generated, which further induce theproliferation of regulatory T cells of patients. The proliferation ofregulatory T cells may suppress immune responses. In addition, thetumor-associated antigens are autoantigens, which may cause autoimmunediseases.

In addition, it has been reported that CD25 monoclonal antibodies can beused to suppress regulatory T cells to improve the effect of immunetherapy. However, the production cost of monoclonal antibodies is high,and the monoclonal antibodies may cause undesired systematicsuppression.

Therefore, it is desirable to provide a drug or an improved method totreat cancer through immune therapy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an RNAi compound or anshRNA expression plasmid, which can encode an siRNA to inhibitexpression of Thrombospondin-1 (TSP-1) through an RNAi pathway. TSP-1regulates immune responses through the activation of functional domainof TGF-β. When the shRNA expression plasmid is used to inhibit theexpression of TSP-1, Dicer cleaves shRNA expression plasmid in cells toform an siRNA, which is a polynucleotide with high specificity. Thestorage of the RNAi compound or the expression plasmid of the presentinvention is very simple. Furthermore, the manufacturing process of theRNAi compound or the expression plasmid of the present invention is muchsimpler than that of protein antigens, so the production cost can begreatly decreased. The RNAi compound or siRNA generated from theexpression plasmid of the present invention can suppress the expressionof the TSP-1 to activate immune responses. If the RNAi compound or siRNAgenerated from the expression plasmid of the present invention can bedelivered into dendritic cells, the immune responses are much moreobvious.

Another object of the present invention is to provide a pharmaceuticalcomposition, which comprises the aforementioned RNAi compound or theaforementioned expression plasmid, and pharmaceutical composition.

A further object of the present invention is to provide a method oftreating cancer, which comprises a step of administering atherapeutically effective amount of the aforementioned pharmaceuticalcomposition.

Another further object of the present invention is to provide a use ofthe aforementioned RNAi compound or the aforementioned plasmid tomanufacture a DNA vaccine.

To achieve the object, the RNAi compound for inhibiting expression ofTSP-1 of the present invention comprises: a target sequence selectedfrom TSP-1 gene.

The expression plasmid for inhibiting expression of TSP-1 of the presentinvention comprises: a target sequence selected from TSP-1 gene; and areverse complement of the target sequence. Herein, the expressionplasmid is an shRNA expression plasmid, and the target sequence encodesan siRNA targeted to TSP-1 gene. In addition, the expression plasmid mayfurther comprise a hairpin sequence locating between the target sequenceand the reverse complement.

In addition, the pharmaceutical composition of the present inventioncomprises: an RNAi compound, and a pharmaceutically acceptable carrier.Herein, the RNAi compound comprises a target sequence selected fromTSP-1 gene.

Furthermore, the method of treating cancer of the present inventioncomprises a step of administering a therapeutically effective amount ofthe aforementioned pharmaceutical composition to a patient in need.

According to the RNAi compound and the expression plasmid of the presentinvention, the target sequence can be any continuous sequence selectedfrom an encoding region corresponding to nucleotides 1 to 3516 of TSP-1gene. Preferably, the target sequence comprises 20-25 nucleotidesselected from an encoding region corresponding to nucleotides 1 to 3516of TSP-1 gene. More preferably, the target sequence is SEQ ID NO: 1, SEQID NO: 2, or SEQ ID NO: 3.

In addition, according to the RNAi compound and the pharmaceuticalcomposition containing the RNAi compound of the present invention, theRNAi compound is an isolated siRNA, or an shRNA expression plasmid forencoding a siRNA. Herein, the siRNA corresponds to the target sequence.Furthermore, the shRNA expression plasmid comprises the target sequence,and a reverse complement of the target sequence, preferably.

According to the present invention, the RNAi compound or the expressionplasmid further comprises: an Her2/neu gene, wherein the Her2/neu geneconnects to the target sequence. Preferably, the sequence of theHer2/neu gene corresponds to nucleotides 21 to 653 of human neu gene.More preferably, the sequence of the Her2/neu gene is SEQ ID NO: 10.

In addition, according to the pharmaceutical composition and the methodof the present invention, the pharmaceutically acceptable carrier isselected from a group consisting of saline, phosphate buffered saline(PBS), and sterile water.

Furthermore, according to the method of the present invention, thepharmaceutical composition is administered through a gene gun or anintramuscular injection.

In conclusion, the present invention provides an RNAi compound and anexpression plasmid for inhibiting expression of TSP-1, a pharmaceuticalcomposition containing the same, and a method of treating cancer byusing the same. Compared to protein antigens conventionally used invaccines, the RNAi compound and the expression plasmid of the presentinvention comprises the advantages of high specificity, fewerside-effects, easy preparation and storage, low production cost, andeasy use. The amount of regulatory T cells can be decreased and thegeneration of cytokines can be suppressed by injection of the RNAicompound once a week. In addition, the survival time of patients can beincreased, and the growth of tumor cells can be delayed.

In addition, the RNAi compound and the expression plasmid of the presentinvention can be used to treat cancer when it is used alone. When theRNAi compound and the expression plasmid is used with other DNAvaccines, the efficiency of DNA vaccines can be greatly increased.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an in vivo experimental process using an shRNA-1 expressionplasmid of the present invention;

FIG. 1B is an in vivo experimental result showing tumor volumes in mice;

FIG. 1C is an in vivo experimental result showing the survivalpercentages of mice, wherein “*” represents p<0.05, “**” representsp<0.01, and “***” represents p<0.005;

FIG. 2A is an in vivo experimental result showing tumor volumes in miceby administering shRNA-1, shRNA-2, and shRNA-3 of the present invention,wherein “*” represents p<0.05, “**” represents p<0.01, and “***”represents p<0.005;

FIG. 2B is an in vivo experimental result showing the survivalpercentages of mice by administering shRNA-1, shRNA-2, and shRNA-3 ofthe present invention;

FIG. 3A is an in vivo experimental result showing tumor volumes in miceby administering shRNA-1 of the present invention combined with a tumorantigen vaccine; and

FIG. 3B is an in vivo experimental result showing the survivalpercentages of mice by administering shRNA-1 of the present inventioncombined with a tumor antigen vaccine, wherein “*” represents p<0.05,“**” represents p<0.01, and “***” represents p<0.005.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

Embodiment Cell Culture

In the present embodiment, mouse bladder tumor cells (MBT-2) and Africangreen monkey kidney fibroblast cells (Cos 7) were cultured in DMEMmedium containing 10% of fetal calf serum (FBS) and 1% ofPenicillin-Streptomycin.

Animal Model

In the present embodiment, female C3H/HeN mice (4-6 weeks of age) wereused in the in vivo experiment.

Plasmid Construction and Vaccine Preparation

The target sequences shown in the following Table 1 (i.e. SEQ ID NOs:1-3) and pHsU6 vectors were used to construct shRNA expression plasmidsof TSP-1. The target sequences were used as forward oligonucleotides,complements of the target sequences were used as reverseoligonucleotides (i.e. SEQ ID NOs: 5-7), and a hairpin sequenceTTCAAGAGA (i.e. SEQ ID NO: 9) was inserted into the target sequences andthe complements. Then, shRNA sequences containing the forwardoligonucleotides, the hairpin sequence, and the reverse oligonucleotideswere inserted into pHsU6 plasmid between the restriction sites of ClaIand HindIII. The obtained pHsU6 shRNA expression plasmids were named asfollow: pHsU6 Tsp-1 shRNA-1, pHsU6 Tsp-1 shRNA-2, and pHsU6 Tsp-1shRNA-3.

TABLE 1 Plasmid Forward/reverse oligonucleotides Tsp-15′-GCCAGAACTCGGTTACCAT-3′ SEQ ID NO: 1 shRNA-1 5′-ATGGTAACCGAGTTCTGGC-3′SEQ ID NO: 5 Tsp-1 5′-CCAACAAACAGGTGTGCAA-3′ SEQ ID NO: 2 shRNA-25′-TTGCACACCTGTTTGTTGG-3′ SEQ ID NO: 6 Tsp-1 5′-GCAACTACCTGGGTCACTA-3′SEQ ID NO: 3 shRNA-3 5′-TAGTGACCCAGGTAGTTGC-3′ SEQ ID NO: 7 Tsp-15′-GGTCCAACAGTCGAACTCT-3′ SEQ ID NO: 4 shRNA-1 5′-AGAGTTCGACTGTTGGACC-3′SEQ ID NO: 8 scrambled shRNA

In addition, U6 promoter in pHsU6 Tsp-1 shRNA-1 plasmid and Tsp-1shRNA-1 gene were inserted into human-cyto-N′-neu plasmid between therestriction sites of AvrII and EcoRI, to obtain a fusion plasmid ofhuman-cyto-N′-neu-Tsp-1 shRNA-1. Herein, the human-cyto-N′-neu plasmidis a plasmid, which contains an encoding region corresponding tonucleotides 21-653 (SEQ ID NO: 10) inserted into pRC/CMV vector(Invitrogen) between NotI and HindIII.

Tsp-1 shRNA-1 scrambled shRNA was prepared by the same method forpreparing Tsp-1 shRNA-1, and used as a negative control. The sequencesof scrambled shRNA were generated with an siRNA Wizard™ programdeveloped by InvivoGen (http://www.sirnawizard.com/). The sequence ofTSP-1 shRNA-1 was used as a template, and re-composed to obtain ascrambled sequence. The obtained sequence of the forward nucleotides andthe reverse nucleotides of the scrambled shRNA were SEQ ID NO: 4 and SEQID NO: 8 respectively, as shown in Table 1.

In addition, the truncated form of Tsp-1 gene (i.e. an encoding regioncorresponding to nucleotides 1083-2642 (SEQ ID NO: 11)) was insertedinto HA6L vector, which was pcDNA3 (Invitrogen) vector connecting withan N-terminal of HA peptide, and then an HA6L Tsp-1 plasmid wasobtained. This obtained HA6L Tsp-1 plasmid was used to generate Tsp-1protein for the following in vitro experiments.

The aforementioned plasmids were purified with Plasmid Mega Kit withoutendotoxin (QIAGEN). The purified shRNA expression plasmid can be used asDNA vaccines in the following experiments.

Tsp-1 RNAi Compound Inhibits Expression of TSP-1 Protein In Vitro.

The transfection was performed with Lipofectamine™ 2000 (Invitrogen).First, DNA was provided, which contained 1.8 μg shRNA and 0.2 μg targetplasmid. The DNA was mixed with Lipofectamine in a ratio of 1:2 at roomtemperature according to the protocol. Then, cells seeded in a plate(3×10⁵/well) were transfected with the mixture. After 24 hrs, proteinswere collected with an RIPA buffer.

The collected proteins were analyzed with a Western Blot Analysisgenerally used in the art. The primary antibody (1:5000 dilution) was amouse anti-HA monoclonal antibody (3F10, Roche), and the secondaryantibody (1:5000 dilution) was a goat anti-mouse IgG (Chemicon). Theinner control of the Western Blot Analysis was actin. The primaryantibody for actin (1:5000 dilution) was an actin antibody (MAB1510,Roche), and the secondary antibody (1:5000 dilution) was an anti-mouseIgG (Cell signally).

The results of the Western Blot Analysis showed that abundant HA Tsp-1fusion protein was generated when HA6L Tsp-1 and pHsU6 plasmid wasco-transfected in a ratio of 1:9 and the total FNA amount was 2 mg.However, when HA6L Tsp-1 and pHsU6 Tsp-1 shRNA-1 plasmid wereco-transfected, the expression of Tsp-1 protein was inhibited by Tsp-1shRNA-1 plasmid.

Tsp-1 RNAi Compound Inhibits Expression of TSP-1 Protein In Vivo.

Tsp-1 shRNA expression plasmid can inhibit expression of Tsp-1 indendritic cells (DCs) inside lymph nodes in vivo.

Saline (control), or saline contained 10 μg of pHsU6 TSP-1 scrambledshRNA-1 plasmid or pHsU6 TSP-1 shRNA-1 plasmid was injected into mouseabdomens with Low-pressure Gene Delivery System (GDS-80, WEALTEC) at 50psi. After 48 hrs, the mice were sacrificed to access inguinal lymphnodes. The lymph nodes were lysed, and CD11c⁺ dendritic cells wereseparated out with CD11c (N418) Microbeads (Miltenyi Biotec). Then, RNAsof CD11c⁺ dendritic cells were obtained with TRIZOL kit (Invitrogen LifeTechnologies), and the RNAs were reverse-transcribed by MMLV reversetranscriptase (Promega) to obtain cDNA. Finally, the expression amountof Tsp-1 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) as aninner control was analyzed with RT-PCR. The primers for Tsp-1 and HPRTare listed as follow.

Primer for Tsp-1 5′-TATGTGCCTAATGCCAACCA-3′ (SEQ ID NO: 12)5′-CGCTGAAGTCCACAGCATTA-3′ (SEQ ID NO: 13)

The results show that the expression of Tsp-1 in dendritic cells (DCs)inside mouse lymph nodes treated with pHsU6 TSP-1 shRNA-1 plasmid of thepresent invention was much lower than that treated with saline and pHsU6TSP-1 scrambled shRNA-1 plasmid. Hence, the TSP-1 shRNA-1 plasmid of thepresent invention can inhibit expression of mRNA of Tsp-1, but the pHsU6TSP-1 scrambled shRNA-1 plasmid having similar composition cannotinhibit expression of mRNA of Tsp-1.

Evaluation of the Effect of Treating MBT-2 Tumor Cells with RNAiCompounds

Mouse bladder tumor cell line MBT-2 with a concentration of 5×10⁶cells/ml PBS was injected into female C3H/HeN mice (4-6 weeks of age)through subcutaneously injection (s.c.), and 1×10⁶ tumor cells wereplanted into dorsum of the mice to perform tumor challenge. The day ofplating tumor cells was Day 1. At Days 8, 15 and 22, the mice weretreated with 10 μg of pHsU6 TSP-1 scrambled shRNA-1 plasmid or pHsU6TSP-1 shRNA-1 plasmid in PBS buffer through a Low-pressure Gene DeliverySystem (GDS-80, WEALTEC) at 50 psi. The schematic outline of theexperimental design is shown in FIG. 1A. The mice treated with salineserved as a control.

After MBT-2 tumor cells were injected, the nodule on the dorsum of themice was measured and the survival percentage was recorded at specifictime points. The equation for calculating the nodule is: V=a²×b×0.5236,wherein V is the volume of tumor, a is the width of tumor, and b is thelength of tumor.

As shown in FIG. 1B, the tumor size on the dorsum of the mice wassmaller than that of the control, and the treating effect of pHsU6 TSP-1shRNA-1 plasmid was better than that of the control. In addition, thesurvival time of the mice treated with pHsU6 TSP-1 shRNA-1 plasmid waslonger than that of the control.

The Expression of Tsp-1 can be Inhibited by shRNA Plasmid with DifferentTsp-1 Fragments In Vitro and In Vivo.

In order to confirm that the shRNA plasmid of the present invention canspecifically inhibit the expression of Tsp-1, TSP-1 shRNA-1 plasmid andshRNA plasmids having two different target sequences were used to testthe effect of the inhibition on Tsp-1.

The transfection and the Western Blot Analysis were performed asillustrated above, except that the shRNA plasmid were pHsU6Tsp-1-shRNA-1, pHsU6 Tsp-1 shRNA-2, pHsU6 Tsp-1 shRNA-3, and pHsU6 TSP-1scrambled shRNA plasmids.

The result of the Western Blot Analysis shows that abundant HA Tsp-1fusion protein was generated when HA6L Tsp-1 and pHsU6 plasmid wasco-transfected. However, when HA6L Tsp-1 was co-transfected with pHsU6Tsp-1 shRNA-1 plasmid, pHsU6 Tsp-1 shRNA-2 plasmid, and pHsU6 Tsp-1shRNA-3 plasmid, the expression of the HA Tsp-1 fusion protein wasinhibited. However, the pHsU6 Tsp-1 scrambled shRNA-1 plasmid, which hassimilar composition to that of pHsU6 Tsp-1 shRNA-1 plasmid, cannotinhibit the expression of Tsp-1 protein.

In addition, the evaluation of the effect of treating MBT-2 tumor cells,which includes measurement of tumor sizes and record of survival time,was performed as illustrated above, except that the shRNA plasmid werepHsU6 Tsp-1 shRNA-1, pHsU6 Tsp-1 shRNA-2, and pHsU6 Tsp-1 shRNA-3plasmids. The results are shown in FIGS. 2A and 2B.

As shown in FIG. 2A, the growth of the tumor cells on the dorsum of themice can be inhibited by administering pHsU6 Tsp-1 shRNA-1, pHsU6 Tsp-1shRNA-2, and pHsU6 Tsp-1 shRNA-3 plasmids of the present invention,compared to that by administering saline (i.e. control). The results ofthe inhibition on tumor cells are consistent with the results of theinhibition on the expression of Tsp-1 protein.

As shown in FIG. 2B, the survival time of the mice administered withpHsU6 Tsp-1 shRNA-1, pHsU6 Tsp-1 shRNA-2, and pHsU6 Tsp-1 shRNA-3plasmids of the present invention were increased, compared to thatadministered with saline (i.e. control).

The Effect of Treating Cancer can be Improved by Administering Tsp-1shRNA-1 Fused with Tumor-Associated Antigen Her2/neu.

The evaluation of the effect of treating MBT-2 tumor cells, whichincludes measurement of tumor sizes and record of survival time, wasperformed as illustrated above, except that shRNA plasmids are pHsU6Tsp-1 shRNA-1 plasmid, human-cyto-N′-neu plasmid, andhuman-cyto-N′-neu-Tsp-1 shRNA-1 plasmid. The results are shown in FIGS.3A and 3B.

As shown in FIG. 3A, the growth of the tumor cells on the dorsum of themice can be inhibited by administering pHsU6 Tsp-1 shRNA-1 plasmid ofthe present invention or human-cyto-N′-neu plasmid (i.e. Her2/neu DNAvaccine), compared to that by administering saline (i.e. control). Inaddition, human-cyto-N′-neu-Tsp-1 shRNA-1 plasmid (i.e. fusion DNAvaccine) showed better inhibition effect on the growth of the tumorcells.

As shown in FIG. 3B, the survival time of the mice administered withpHsU6 Tsp-1 shRNA-1 plasmid or human-cyto-N′-neu plasmid (i.e. Her2/neuDNA vaccine) were increased, compared to that administered with saline(i.e. control). In addition, human-cyto-N′-neu-Tsp-1 shRNA-1 plasmid(i.e. fusion DNA vaccine) showed better effect on the increase of thesurvival time.

In conclusion, according to the in vivo experiment, the expression ofTsp-1 in dendritic cells (DCs) inside lymph nodes can be inhibitedthrough administering the Tsp-1 shRNA plasmid of the present inventionwith a gene gun. In addition, according to the experiment on mice withbladder tumor cells, the Tsp-1 shRNA plasmid with specificity of thepresent invention can significantly inhibit the growth of tumor cells,and extend the survival time of mice.

In addition, the Tsp-1 shRNA plasmids with different Tsp-1 fragmentshave similar therapeutic effects. Hence, the Tsp-1 shRNA plasmids of thepresent invention can specifically inhibit the expression of Tsp-1protein.

When the Tsp-1 shRNA is fused with tumor-associated antigen Her2/neugene, the effect of DNA vaccine can be further improved. Hence, theTsp-1 shRNA indeed can improve anticancer immune responses. Therefore,the siRNA compound with Tsp-1 gene can trigger the anticancer immuneresponses, and can be used as DNA vaccine or be used with other immunetherapies.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thescope of the invention as hereinafter claimed.

1. An RNAi compound for inhibiting expression of Thrombospondin-1,comprising: a target sequence selected from Thrombospondin-1 gene. 2.The RNAi compound as claimed in claim 1, wherein the target sequencecomprises 20-25 nucleotides, and is selected from an encoding regioncorresponding to nucleotides 1 to 3516 of Thrombospondin-1 gene.
 3. TheRNAi compound as claimed in claim 1, wherein the target sequence is SEQID NO: 1, SEQ ID NO: 2, or SEQ ID NO:
 3. 4. The RNAi compound as claimedin claim 1, further comprising an Her2/neu gene, wherein the Her2/neugene connects to the target sequence.
 5. The RNAi compound as claimed inclaim 4, wherein the sequence of the Her2/neu gene is SEQ ID NO:
 10. 6.The RNAi compound as claimed in claim 1, wherein the RNAi compound is anisolated siRNA, or an shRNA expression plasmid for encoding an siRNA. 7.The RNAi compound as claimed in claim 6, wherein the shRNA expressionplasmid comprises the target sequence, and a reverse complement of thetarget sequence.
 8. An expression plasmid for inhibiting expression ofThrombospondin-1, comprising: a target sequence selected fromThrombospondin-1 gene; and a reverse complement of the target sequence,wherein the target sequence encodes an siRNA targeted toThrombospondin-1 gene.
 9. The expression plasmid as claimed in claim 8,wherein the target sequence comprises 20-25 nucleotides, and is selectedfrom an encoding region corresponding to nucleotides 1 to 3516 ofThrombospondin-1 gene.
 10. The expression plasmid as claimed in claim 8,wherein the target sequence is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3.
 11. The expression plasmid as claimed in claim 8, further comprisingan Her2/neu gene, wherein the Her2/neu gene connects to the targetsequence.
 12. The expression plasmid as claimed in claim 11, wherein thesequence of the Her2/neu gene is SEQ ID NO:
 10. 13. A pharmaceuticalcomposition, comprising: an RNAi compound, which comprises a targetsequence selected from Thrombospondin-1 gene; and a pharmaceuticallyacceptable carrier.
 14. The pharmaceutical composition as claimed inclaim 13, wherein the target sequence comprises 20-25 nucleotides, andis selected from an encoding region corresponding to nucleotides 1 to3516 of Thrombospondin-1 gene.
 15. The pharmaceutical composition asclaimed in claim 13, wherein the target sequence is SEQ ID NO: 1, SEQ IDNO: 2, or SEQ ID NO:
 3. 16. The pharmaceutical composition as claimed inclaim 13, wherein the RNAi compound further comprises an Her2/neu gene,wherein the Her2/neu gene connects to the target sequence.
 17. Thepharmaceutical composition as claimed in claim 16, wherein the sequenceof the Her2/neu gene is SEQ ID NO:
 10. 18. The pharmaceuticalcomposition as claimed in claim 13, wherein the RNAi compound is anisolated siRNA, or an shRNA expression plasmid for encoding an siRNA.19. The pharmaceutical composition as claimed in claim 18, wherein theshRNA expression plasmid comprises the target sequence, and a reversecomplement of the target sequence.
 20. The pharmaceutical composition asclaimed in claim 13, wherein the pharmaceutically acceptable carrier isselected from a group consisting of saline, phosphate buffered saline(PBS), and sterile water.
 21. A method of treating cancer, comprising:administering a therapeutically effective amount of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises: an RNAicompound, which comprises a target sequence selected fromThrombospondin-1 gene; and a pharmaceutically acceptable carrier. 22.The method as claimed in claim 21, wherein the target sequence comprises20-25 nucleotides, and is selected from an encoding region correspondingto nucleotides 1 to 3516 of Thrombospondin-1 gene.
 23. The method asclaimed in claim 21, wherein the target sequence is SEQ ID NO: 1, SEQ IDNO: 2, or SEQ ID NO:
 3. 24. The method as claimed in claim 21, whereinthe RNAi compound further comprises an Her2/neu gene, wherein theHer2/neu gene connects to the target sequence.
 25. The method as claimedin claim 24, wherein the sequence of the Her2/neu gene is SEQ ID NO: 10.26. The method as claimed in claim 21, wherein the RNAi compound is anisolated siRNA, or an shRNA expression plasmid for encoding an siRNA.27. The method as claimed in claim 26, wherein the shRNA expressionplasmid comprises the target sequence, and a reverse complement of thetarget sequence.
 28. The method as claimed in claim 21, wherein thepharmaceutically acceptable carrier is selected from a group consistingof saline, phosphate buffered saline (PBS), and sterile water.
 29. Themethod as claimed in claim 21, wherein the pharmaceutical composition isadministered through a gene gun or an intramuscular injection.